Our long-term goal is to elucidate the molecular mechanisms of regulated Cu efflux by two mammalian Cu- ATPases in polarized epithelial cells. ATP7A in intestinal epithelial cells delivers Cu to the circulation (basolateral environment) and ATP7B in hepatic cells delivers Cu to the bile (apical environment). Mutations in either protein cause human disease, emphasizing their importance in Cu-homeostasis. Based on extensive preliminary data in vivo and in vitro, we propose to test three hypotheses. In AIM 1 we hypothesize that in Cu-loaded hepatic and intestinal cells, Cu-ATPases pump Cu into unique vesicles that fuse with the basolateral (ATP7A) or apical (ATP7B) plasma membrane (PM) to release "stored" Cu. We will identify these compartments using immuno-EM and immuno-isolation/proteomic approaches. In AIM 2 we hypothesize that the chloride channel, CIC4, is an anion shunt for both Cu-ATPases in their secretory and efflux function/locations. In basal Cu, We will determine if Cu loading of ceruloplasmin in WIF-B cells by ATP7B and hephaestin in Caco-2 cells by ATP7A occurs in chloride-free conditions and after knock-down of CIC4. We will also examine the role of CIC4 in Cu efflux from the cells.In Aim 3, we hypothesize that when Cu levels are elevated, unique structural signals in the N-terminus of ATP7B regulate its trafficking/apical efflux function. Our test includes biochemically isolating and identifying new protein interactors, elucidating the role of a newly-identified modulator of Cu efflux (Murr1) on the dynamics/function of wt and selected N-terminal mutants of exogenous ATP7B, and using the LEG rat model of Wilson Disease to confirm and extend in vitro findings. Approaches will include RNAi and overexpression of wt modulators, as well as yeast two hybrid. Also in AIM 2 we hypothesize that when Cu levels are lowered, ATP7A and ATP7B are retrieved from their distinct compartments through recognition of structural signals in their C-termini. We will use yeast two hybrid and affinity isolation of putative protein interactors from extracts of relevant tissue.